Field of the Invention
The subject of the invention is a method for purifying the rabies virus that has been obtained from a culture of cells.
Summary of the Related Art
In general, virus harvests obtained from infected cell cultures contain not only the desired viruses, but also the proteins and the DNA of the cells, which are impurities which should be removed. The amounts of cellular proteins and DNA released are all the greater if the viruses are responsible for considerable cell lysis and/or if the viral harvests are carried out late. In addition to the impurities of cellular nature, the proteins of the medium containing the infected cells are also impurities that are also intended to be removed during the implementation of the viral purification process.
When the viruses are used to manufacture vaccines, it is advisable to obtain preparations which are as pure as possible so as to prevent the development of allergic reactions against the protein impurities. There are also countries that limit the maximum authorized amount of cellular DNA in vaccines comprising products obtained from continuous cell lines to 100 pg/vaccine dose or even less.
Various methods for purifying the rabies virus have already been described in the prior art:
U.S. Pat. No. 4,664,912 describes a method for purifying the rabies virus by zonal centrifugation after it has been inactivated with β-propiolactone. Another method consists in combining size exclusion chromatography and anion exchange chromatography when the volume of the harvest of virus to be purified is large.
U.S. Pat. No. 4,725,547 describes a method for purifying the rabies virus by affinity chromatography on cellulofine sulfate (a sulfuric acid ester of cellulose).
WO 97/06243 describes a method for purifying viruses, in particular the rabies virus, from a culture of infected Vero cells. The method comprises anion exchange chromatography followed by cation exchange chromatography and is completed by metal-binding affinity chromatography. Using this method, the amount of residual DNA contained in one vaccine dose is ≦30-40 pg, using the “Threshold Total DNA assay” technique.
Kumar A. P et al., in Microbes and Infection (2005) 7; 1110-1116, have compared two methods for purifying the rabies virus from a culture supernatant of infected Vero cells that were cultured in a medium containing fetal calf serum. It shows that the purification method based on the use of an anion exchange chromatography column (DEAE-sepharose CL-6B) is more effective than the method of purification by zonal centrifugation on a sucrose gradient, since, at a comparable degree of purity in terms of amount of residual proteins and nucleic acids, the rabies virus yield is better with the chromatographic method. Frazatti-Gallina N. M., in Vaccine (2004) 23; 511-517, has also described a method for purifying the rabies virus from a culture supernatant of infected Vero cells that were cultured in a medium free of fetal calf serum. The purification method also implements a step of anion exchange chromatography on a DEAE-cellulose-based support, after a step of clarifying and concentrating the viral harvest. Using this method, the amount of residual DNA measured by slot/blot hybridization technique is <23 pg per vaccine dose.
Thus, when the method for purifying the rabies virus comprises an ion exchange chromatography step, there is virtually systematically an anion exchange chromatography step so as to retain the nucleic acids of the medium containing the virus to be purified, on the chromatographic support.